Fatty acids containing two or more unsaturated bonds are collectively referred to as polyunsaturated fatty acids (PUFAs) and are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. Some of these polyunsaturated fatty acids cannot be synthesized in the animal body, and such polyunsaturated fatty acids need to be ingested from foods as essential fatty acids. The polyunsaturated fatty acids are widely distributed. For example, arachidonic acid is isolated from lipids extracted from suprarenal gland and liver of animals. However, the amounts of these polyunsaturated fatty acids contained in animal organs are small, and the polyunsaturated fatty acids extracted and isolated from animal organs only are insufficient for a large amount of supply thereof. Thus, microbial techniques have been developed for obtaining polyunsaturated fatty acids by culturing various microorganisms. In particular, microorganisms in the genera Mortierella are known to produce lipids containing polyunsaturated fatty acids such as arachidonic acid.
Other attempts have also been made to produce polyunsaturated fatty acids in plants. Polyunsaturated fatty acids are known to constitute reserve lipids such as triacylglycerol (also referred to as triglyceride or TG) and accumulate within microorganism cells or plant seeds.
Triacylglycerol as a reserve lipid is generated in the body as follows: An acyl group is introduced into glycerol-3-phosphate by glycerol-3-phosphate acyltransferase to generate lysophosphatidic acid. An acyl group is introduced into the lysophosphatidic acid by lysophosphate acyltransferase to generate phosphatidic acid. The phosphatidic acid is dephosphorylated by phosphatidic acid phosphatase to generate diacylglycerol. An acyl group is introduced into the diacylglycerol by diacylglycerol acyltransferase to generate triacylglycerol.
In this pathway, phosphatidic acid (hereinafter, also referred to as “PA” or 1,2-diacyl-sn-glycerol-3-phosphate) is a precursor of triacylglycerol and is also a biosynthetic precursor of diacyl glycerophospholipid. In yeast cells, CDP diacylglycerol (CDP-DG) is synthesized from PA and cytidine 5′-triphosphate (CTP), by phosphatidate cytidyltransferase, and is biosynthesized into various phospholipids.
As described above, the reaction of biosynthesizing diacylglycerol (hereinafter, also referred to as “DG”) through dephosphorylation of PA is known to be catalyzed by phosphatidic acid phosphatase (E.C. 3.1.3.4, hereinafter, also referred to as “PAP”). This PAP is known to be present in all organisms from bacteria to vertebrates.
Yeast (Saccharomyces cerevisiae), which is a fungus, has two types of PAPs (Non-Patent Literatures 1, 2, and 7). One is a Mg2+-dependent PAP (PAP1), and the other is a Mg2+-independent PAP (PAP2). A PAH1 gene is known as a gene encoding PAP1 (Non-Patent Literatures 3 to 5). A pah1Δ variant also shows a PAP1 activity, which suggests there are other genes exhibiting the PAP1 activity. In the pah1Δ variant, the nuclear membrane and the ER membrane are abnormally dilated, and expression of important genes for biosynthesis of phospholipids is abnormally enhanced (Non-Patent Literature 6).
As genes encoding PAP2, a DPP1 gene and a LPP1 gene are known and exhibit most PAP2 activities in yeast. The enzymes encoded by these genes have broad substrate specificity and act also on, for example, diacylglycerol pyrophosphate (DGPP), lysophosphatidic acid, sphingoid base phosphate, and isoprenoid phosphate to dephosphorylate them.
A lipid-producing fungus, Mortierella alpina, is known to have a MaPAP1 gene, which is a Mg2+-independent PAP2 homolog (Patent Literature 1).
Existance of gene homologs that probably encode PAP1 family enzymes or PAP2 family enzymes in other bacteria is known in the art, but their functions have not been elucidated.